Project Summary/Abstract Abnormalities in phosphosignaling networks in cancers are often the direct or indirect targets of anti-tumor agents. Thus it is critical that we are able to reliably quantify phosphoproteins in cancer biopsies to establish mechanism of action, to evaluate pharmacodynamic responses (to inform dose and scheduling), and to monitor compensatory pathways that influence efficacy and toxicity. Prior studies have shown that a significant subset of the cancer phosphoproteome is highly susceptible to preanalytical variables, such as ischemic time prior to fixation or freezing. Thus, it is critical that the collection and processing of cancer biopsies be conducted using a strict SOP that minimizes preanalytical variations. For extraction-based methods (e.g. mass spectrometry, ELISA, Western blotting), the current gold standard is to flash freeze tumor samples in liquid nitrogen, followed by preparation of protein lysates in denaturing conditions in the presence of kinase/phosphatase inhibitor cocktails. However, a major limitation is that in many clinical settings liquid nitrogen is not readily available, and neither the personnel nor the infrastructure is generally available to rapidly process the tumor samples. As a result, tumors are often subjected to prolonged ischemia and/or chemical fixatives, altering the phosphoproteome and thereby compromising the biospecimen?s integrity such that it may no longer reflect the true in vivo state of the tumor. We propose to develop an affordable, convenient, single-use device for rapidly freezing tumor biopsies in clinical settings to preserve the labile phosphoproteome for molecular analyses.